DESCRIPTION: Adenovirus vectors (Ad) are the second most frequently used vectors in clinical trials in the US to treat numerous inborn and acquired human diseases. Even though natural infections with wild-type Ad are largely harmless in humans, an intravenous Ad administration for gene delivery purposes, especially at high doses, stimulates strong innate immune and inflammatory host responses. Using an entirely in vivo approach and a large collection of gene-deficient mouse models, the PI's laboratory has discovered that Ad vectors in the blood bind coagulation factors that facilitate virus recognition by innate immune receptors and activate IL-1 family cytokines, which, in turn, trigger a cascade of cellular and molecular effectors of innate immunity and systemic inflammation. Most recently, we found that Ad administration triggers a novel necrosis-like type of cell death of liver residential macrophages, Kupffer cells, that is mechanistically controlled by interferon regulatory factor 3, IRF3. Importantly, in addition to Ad vectors, this rapid necrotic Kupffer cell death was also observed after intravascular delivery into mice of both the bacterial pathogen L.monocytogenes and fully synthetic nanoparticles. Our preliminary studies in animal models of disseminated infections revealed that IRF3- dependent macrophage necrosis is highly pro-inflammatory and contributes to leukocytopenia, a clinical sign of systemic inflammation. The major goals of this grant proposal are i) to define the molecular mechanism and signaling partners that activate IRF3 to execute necrotic macrophage cell death in vivo and ii) to define the innate immune and inflammatory responses to gene transfer vectors in a novel humanized mouse model. These goals will be achieved via the following Specific Aims: 1. To identify the molecular signature of IRF3 modification(s) associated with its function as a trigger of necrotic Kupffer cell death in response to Ad in vivo. 2. To identify components of the signaling pathway that target IRF3 for execution of necrotic Kupffer cell death in vivo. 3. To define the signature of innate immune activation and systemic innate immune and inflammatory responses to gene transfer vectors in a novel humanized mouse model. The conceptual and mechanistic advances obtained from the proposed studies have the potential to greatly improve our understanding of Ad interactions with host cells in vivo, provide new insights into fundamental cell and molecular biological processes related to pro-inflammatory anti-pathogen responses, and will allow for optimization of gene transfer vectors for vaccination and gene transfer applications.